1,2,4-Benzothiadiazine linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates: Synthesis, DNA-binding affinity and cytotoxicity

Article abstract of DOI:10.1016/j.bmcl.2007.08.018

Benzothiadiazine-pyrrolobenzodiazepine conjugates linked through different alkane spacers have been prepared. These new classes of hybrid molecules exhibit cytotoxicity against many cancer cell lines. Their DNA thermal denaturation studies have been carri

Full text of DOI:10.1016/j.bmcl.2007.08.018

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5345–5348
1,2,4-Benzothiadiazine linked pyrrolo[2,1-c][1,4]benzodiazepine
conjugates: Synthesis, DNA-binding affinity and cytotoxicity
Ahmed Kamal,a,* M. Naseer A. Khan,a K. Srinivasa Reddy,a S. Kaleem Ahmed,a
M. Shiva Kumar,a Aarti Juvekar,b Subrata Senb and Surekha Zingdeb
aBiotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India
bAdvanced Centre for Treatment, Research & Education in Cancer, Navi Mumbai, India
Received 18 April 2007; revised 8 August 2007; accepted 8 August 2007
Available online 15 August 2007
Abstract—Benzothiadiazine–pyrrolobenzodiazepine conjugates linked through different alkane spacers have been prepared. These
new classes of hybrid molecules exhibit cytotoxicity against many cancer cell lines. Their DNA thermal denaturation studies have
been carried out and one of the compounds (4b) elevates the DNA helix melting temperature of the CT-DNA by 6.7 °C after incu-
bation for 36 h.
Ó 2007 Elsevier Ltd. All rights reserved.
In recent years, combination chemotherapy with different
mechanisms of action is one of the methods that is being
adopted to treat cancer. Therefore, a single molecule con-
taining more than one pharmacophore, each with differ-
ent mode of action, could be beneficial for the treatment
of cancer. Sulfonylurea derivatives constitute an impor-
tant class of therapeutic agents in medicinal chemistry.1
Compound LY186641 (1) was reported to possess a
broad spectrum of activity in several solid tumour mod-
els,2–4 and reached the clinical trials based on its impres-
sive preclinical activity and apparent lack of toxicity to
proliferating normal tissues.5,6 The mode of action of
these compounds differs from the traditional anticancer
drugs which typically inhibit DNA, RNA, or protein syn-
thesis. Further, it was found to accumulate in the cell
mitochondria which may be target site for antitumour
activity of these compounds.7,8 1,2,4-Benzothiadiazine
1,1-dioxide ring system and 2,10-dihydro-10-hydroxy-
3H-imidazo[1,2-b][1,2,4]benzothiadiazine 6,6-dioxide
(2) contain a built-in sulfonylhydroxyguanidine moiety.
Compound 2 exhibits its activity against several tumour
cell lines, which is considered to combine the imino group
of guanidine along with the hydroxylamino group of
hydroxyurea. The potent antiviral and anticancer activi-
ties of this compound are exhibited by inhibition of ribo-
nucleotide reductase.9,10
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a
group of naturally occurring antitumour antibiotics,
members of which include anthramycin, tomamycin,
neothramycins A and B, sibiromycin, mazethramycin,
chicamycin, prothracarin, DC-81 (3) and dextochry-
sin.11 The formation of a covalent bond in the minor
groove of DNA by nucleophilic attack of 2-amino group
of guanine base to form an amino linkage to C-11 is
responsible for the biological activities of PBDs.12 In
the past few years, several hybrid compounds, in which
known antitumour agents tethered to PBD moiety, have
been designed, synthesized and evaluated for their bio-
logical activity.13–15 Recently, we have been involved
in the development of new synthetic strategies16 for
the preparation of PBD ring system and also in the de-
sign of structurally modified PBDs and their hybrids for
the development of more potent anticancer agents.17
A number of piperazine derivatives have been synthe-
sized for their chemotherapeutic use in the area of
medicinal chemistry.18 Michejda and co-workers19 re-
ported symmetrical bifunctional agents as a promising
antitumour class of compounds with remarkable selec-
tivity against colon cancers that possess a piperazine
moiety in its linker spacer. Recently, trans-diamine
dichloroplatinum(II) complexes with piperazine ligands
have exhibited significant cytotoxicity.20 In these, plati-
num–piperazine complexes are taken up by the cancer
Keywords: Benzothiadiazines; Pyrrolobenzodiazepines; Cytotoxicity;
DNA binding.
*
Corresponding author. Tel.: +91 40 27193157; fax: +91 40
27193189; e-mail: ahmedkamal@iict.res.in
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.018

5346
A. Kamal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5345–5348
cells and bind to DNA much faster than the cisplatin.
Moreover, some of the PBD conjugates with piperazinyl
alkane spacers have also shown promising anticancer
activity.21
O
O
S
O
O
NH
S
N
N
HN
O
N
OH
The 1,2,4-benzothiadiazine has been linked through 3-
piperazinyl alkane spacer to the C8-position of the A
ring of PBD ring system to explore their potential as
new class of anticancer agents. Such conjugates with
the combination of DNA binding and DNA, RNA or
protein synthesis inhibitors could be attractive targets
for their DNA-binding potential as well as antitumour
activity (Fig. 1).
Cl
2
(1, LY-186641)
O
O
S
N
N
N
H
HO
N
N
N
O
H
( )4
N
O
H3CO
Synthesis of these benzothiadiazine–pyrrolobenzodiaze-
pine conjugates has been carried out by employing 3-
chloro-1,2,4-benzothiadiazines (8a–b), which have been
obtained by previously reported methods22 and the pre-
cursors 5a–b have also been prepared by the methods
reported in our earlier studies.17 These precursors have
been treated with N-Boc piperazine to give 6a–b, which
on Boc deprotection yield intermediates 7a–b. 10-Substi-
tuted 3-chloro-1,2,4-benzothiadiazines (8a–b) are then
coupled with 7a–b in the presence of triethylamine to
N
H3CO
O
4c
(3, DC-81)
Fig. 1. Chemical structures of sulfonylurea derivative (1, LY-186641),
imidazo benzothiadiazine derivative (2), DC-81 (3) and benzothiadi-
azine–PBD conjugate (4c).
afford 9a–b, which upon reduction and cyclisation give
23
the target molecules 4a–d (Scheme 1).
O
NO2
CH(SEt)2
O
NO2 CH(SEt)2
Br (H2C)n
Boc
N
N
(H2C)n
H3CO
6a-b
i
N
O
N
O
H3CO
5a-b
ii
O
O
O
NO2
CH(SEt)2
S
HN
N
(H2C)n
N
N
H3CO
N
R
Cl
O
8a-b
7a-b
iii
R = (CH3)2CH, C6H5
n = 4, 5
O
O
N
S
O
NO2
CH(SEt)2
N
(H2C)n
N
R
N
N
H3CO
O
9a-d
iv, v
O
O
S
N
N
R
N
4a : R = (CH3)2CH, n = 4
4b : R = (CH3)2CH, n = 5
4c : R = C6H5, n = 4
N
O
N
O
H
( )n
N
H3CO
4d : R = C6H5, n = 5
4a-d
Scheme 1. Reagents and conditions: (i) N-Boc-piperazine, K2CO3, acetone, reflux, 24 h, 85–90%; (ii) TFA, CH2Cl2, 0 °C, 8 h, 75–80%; (iii) THF,
Et3N, rt, 6–8 h, 75–80%; (iv) SnCl2Æ2H2O, MeOH, 4 h, reflux; (v) HgCl2, CaCO3, CH3CN/H2O, 12 h, rt, 55–60%.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5345–5348
Table 1. IC50 valuesa (in lM) for compounds 4a–d in selected human cancer cell lines
5347
Compound
A549b
Hop62b
Zr-75-1c
DWDd
KBd
Guravd
PC3e
f
f
f
f
4a
4b
—
30.30
343.00
27.90
369.00
2.24
0.12
0.15
—
27.7
26.10
2.43
—
—
26.50
f
—
356.00
0.16
27.80
357.00
2.35
f
f
4c
4d
—
30.20
1.92
—
29.40
2.30
0.17
f
f
f
—
f
—
2.32
—
24.90
ADR
—
2.26
a Mean of three determinations.
b Lung cancer.
c Breast cancer.
d Oral cancer.
e Prostate cancer.
f IC50 value not attained at the concentrations used in the assay; ADR, adriamycin.
Control
UC
Concentration (μM)
Compounds 4a–d have been evaluated for their in vitro
cytotoxicity in selected human cancer cell lines of lung,
breast, oral and prostate by using Sulforhodamine
5
C
10 15 20
25 30
(SRB) method.24,25 The compounds exhibiting IC50
6
10À5 M are considered to be active on the respective cell
lines. Table 1 reveals that compounds 4a–c have exhib-
ited strong effect against Zr-75-1 cell line (IC50 0.12–
2.24 lM) in comparison to adriamycin (IC50 2.32 lM).
The in vitro cytotoxicity (IC50) for compound 4b is
2.43 lM in KB cell lines and in case of 4d, the activity
is 2.30 and 2.35 lM in KB and PC3 cell lines, respec-
tively, whereas, this value for adriamycin is 0.17 and
2.26 lM for the same cell lines. Therefore, the in vitro
cytotoxicity exhibited by these new PBD–benzothiadi-
azine conjugates is highly significant.
Fig. 2. RED100-restriction endonuclease digestion assay for a C8-
linked PBD with CT-DNA inhibitory activity of 4c on the cleavage of
plasmid pBR322 by restriction endonuclease BamH1 (20 U in 2 lL)
for 1 h at 37 °C. The cut (C) and uncut (UC) products were separated
by agarose gel electrophoresis and visualized by ethidium bromide
staining under UV illumination. Lane 1: control pBR322; lane 2:
complete digest of pBR322 by BamH1; lanes 3–8: increasing concen-
tration of 4c.
The DNA-binding ability for these benzothiadiazine–
PBD conjugates has been determined by thermal
denaturation studies using calf thymus (CT)-DNA.
These studies were carried out at PBD/DNA molar ra-
tio 1:5. Interestingly, all the compounds (4a–d) elevate
the helix melting temperature of CT-DNA in the
range of 3.9–6.1 °C at 0 h and also examined after
18 and 36 h incubation at 37 °C. Compound 4b
showed highest DTm of 6.1 °C at 0 h and increased
up to 6.7 °C after 36 h incubation, whereas the natu-
rally occurring DC-81 (1) exhibits a DTm of 0.7 °C
after incubation under similar conditions (Table 2).
This result indicates the effect on DNA-binding affin-
ity introducing the benzothiadiazine moiety to PBD
through piperazine with different alkane piperazine
spacers at C8-position of the DC-81 possesses good
DNA-binding ability.
The restriction endonuclease inhibition assay carried out
on these molecules also confirms the relative binding
affinity of these PBD conjugates. The experimental proto-
col is described in the previous study.26,27 The result of a
representative compound 4c has been shown in Figure 2,
suggesting the inhibition of BamH1 by this PBD hybrid.
Table 2. Thermal denaturation data for benzothiadiazine–PBD con-
jugates with calf thymus (CT) DNA
Compound
[PBD]:[DNA] molar ratiob
DTm (°C)a after
In conclusion, these new synthesized benzothiadiazine–
PBD conjugates have exhibited significant in vitro antit-
umour activity against breast, oral and prostate human
cancer cell lines and remarkable DNA-binding ability.
Further, the detailed biological and molecular modelling
studies are in progress.
incubation at 37 °C
0 h
18 h
36 h
4a
4b
1:5
1:5
1:5
1:5
1:5
3.9
6.1
4.0
4.2
0.3
4.2
6.4
4.1
4.4
0.7
4.3
6.7
4.1
4.6
0.7
4c
4d
DC-81
a For CT-DNA alone at pH 7.00 0.01, Tm = 69.2 °C 0.01 (mean
value from 10 separate determinations), all DTm values are 0.1–
0.2 °C.
Acknowledgments
b For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentra-
tion is 100 lM and ligand concentration is 20 lM in aqueous sodium
phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH
7.00 0.01].
The authors M.N.A., K.S.R. and S.K.A. thank CSIR,
New Delhi, for the award of research fellowships and
A.K., A.S.J. and S.M.Z. thank DBT for funding the
project.

5348
A. Kamal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5345–5348
16. (a) Kamal, A.; Laxman, E.; Reddy, P. S. M. M.
Supplementary data
Tetrahedron Lett. 2000, 41, 7743; (b) Kamal, A.;
Reddy, G. S. K.; Raghavan, S. Bioorg. Med. Chem.
Lett. 2001, 13, 387; (c) Kamal, A.; Reddy, P. S.
M. M.; Reddy, D. R. Tetrahedron Lett. 2003, 44,
2557.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.08.018.
17. (a) Kamal, A.; Laxman, E.; Laxman, N.; Rao, M. V.
Bioorg. Med. Chem. Lett. 2000, 10, 2311; (b) Kamal,
A.; Ramesh, G.; Laxman, N.; Ramulu, P.; Srinivas,
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2928, 2866, 1602, 1532, 1451, 1304, 1255, 1146 (SO2), 877.
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J = 7.62 Hz, 1H), 7.65 (d, J = 5.08 Hz, 1H), 7.28–7.58 (m,
10H), 6.77 (s, 1H), 4.02 (d, J = 5.92 Hz, 1H), 3.90 (s, 3H),
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J = 6.77 Hz, 2H). FAB-MS m/z 643 (M+1)+; IR (KBr)
(mmax/cmÀ1): 2928, 1597, 1547, 1428, 1301, 1263, 1164
(SO2), 759. The detail spectral data of other compounds
and experimental procedures are available in Supplemen-
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